RNA splicing, in molecular biology, is a form of RNA processing in which a newly made precursor messenger RNA transcript is transformed into a mature messenger RNA. During splicing, introns are removed and exons are joined together.
2. RNA Processing
Post-transcriptional Processing or RNA processing is a set of
biological processes common to mosteukaryotic cells by which
anRNA primary transcript is chemically altered following
transcription from a gene to produce a mature, functional RNA
molecule that can then leave the nucleus and perform any of a
variety of different functions in the cell. There are many types of
post-transcriptional modifications achieved through a diverse
class of molecular mechanisms. Phillip Sharp and RichardRoberts
were awarded the 1993 Nobel Prize in Physiology or Medicine for
their discovery of introns and the splicing process
3.
4. • In prokaryotes, no RNA processing is necessary: – the
nascent RNA is usually the mRNA.
• In eukaryotes, the nascent RNA is called primary
transcript-RNA – needs to be processed – and transported
to the cytoplasm for translation to occur.
• The processing steps are:
– Addition of a 5’ 7-methyl guanosine cap (capping).
– Addition of a poly-A tail at the 3’ end (polyadenylation)
– RNA splicing to remove intervening sequences (remove
introns
5.
6.
7. When the RNA chain is about 30 nucleotides long, the 5’ ends are
modified by the addition of a guanine group in the opposite
orientation:
– involves a 5’-5’ triphosphate linkage
10. Eukaryotic RNA Processing:Polyadenylation
• nascent RNA is cleaved downstream from the
AAUAAA
conserved sequence.
– By ribonuclease
• The enzyme poly(A) polymerase adds adenine
ribonucleotides
– up to 200 bases long at the 3’ end of the RNA.
• The poly(A) tail
– enhances the stability of eukaryotic mRNA and
– regulates its transport to the cytoplasmic
compartment
TAILING
11. Polyadenylation: The Proteins
Proteins required in mammals for cleavage
and polyadenylation of a new transcript.
Proteins required for efficient cleavage of pre-mRNA:
1. CPSF (cleavage & polyadenylation specificity factor), binds the
AAUAAA
2. CstF (cleavage stimulation factor) binds to the G/U rich region
cooperatively with CPSF
3. CFI and CFII (cleavage factors I and II), RNA-binding proteins
4. PAP (polyA polymerase)
5. nRNAP II (the CTD of the very large RPB1 subunit)
stimulates cleavage
17. The Spliceosome
A spliceosome is a large and complex molecular machine found primarily within
the nucleus of eukaryotic cells.The spliceosome is assembled from small nuclear
RNAs (snRNA) and approximately 80 proteins.
snRNAs (U1, U2, U4, U5 and U6) and associated proteins = snRNPs
• U1 binds to the GU sequence at the 5' splice site, along with accessory
proteins/enzymes,
• U2 binds to the branch site, and ATP is hydrolyzed;
• U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to
U2;
• U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site;
• U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site,
and the 5' site is cleaved, resulting in the formation of the lariat;
• U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are
ligated usingATP hydrolysis.The spliced RNA is released and the lariat
debranches.
18. Three types of short sequences dictate the precise cutting
of the
intron/exon boundaries - called splice junctions.
– Splice donor: 5’ end of intron: exon-G-U
– SpliceAcceptor: 3’ end of intron: A-G-exon
– Branch site: within the intron, about 30 nucleotides
upstream of the
splice acceptor, has an AT rich region with at least one A.
